Genotoxic potential of cyclosporin A in patients with renal transplantation

We analyzed the induction of sister chromatid exchange (SCE) by cyclosporin A (CsA) as a marker of genotoxic potential. In 30 patients undergoing renal transplantation, SCE induction was tested before the introduction of CsA and 3 months later. We found that SCE frequency increased significantly at the end of 3 months. To our knowledge, this is the first study demonstrating in vivo induction of SCE by CsA in humans. We conclude that CsA has a genotoxic potential on human lymphocytes.     

Comparison of long-term survival of pigmented epidermal reconstructs cultured in vitro vs. xenografted on nude mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2-3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long-term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long-term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long-term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan-A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

Dimethyl Sulfoxide and Ebselen Prevent Convulsions Induced by 5-Aminolevulinic Acid

We investigated whether intrastriatal (i.s.) administration of 5-aminolevulinic acid (ALA) induces oxidative damage and whether behavioral alterations induced by i.s. administration of ALA could be affected by antioxidants. Unilateral injection of ALA (6 micromol/striatum) increased (approximately 30%) thiobarbituric acid-reactive substances (TBARS), but did not affect striatal content of total thiol groups. ALA-induced body asymmetry was not prevented by pretreatment with ascorbic acid (100 mg/kg, s.c.), dimethyl sulfoxide (DMSO, 0.5 microl/striatum, i.s.) or ebselen (10 nmol/striatum, i.s.). ALA-induced convulsions were not prevented by ascorbic acid, but were partially prevented by DMSO and completely prevented by ebselen. Ebselen completely prevented the increase of striatal TBARS induced by ALA. Results obtained suggest the involvement of reactive species in ALA-induced convulsions and may be of value in understanding the physiopathology of neurological dysfunctions associated to ALA overload.     

Three acylated flavone glycosides from Sideritis ozturkii Aytac & Aksoy

From the aerial parts of Sideritis ozturkii, three new flavonoids, chrysoeriol 7-O-[2'-O-caffeoyl-O-acetyl-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside], chrysoeriol 7-O[2'-O-caffeoyl-beta-D-glucopyranosyl-(1 -->2)-beta-D-glucopyranoside] and chrysoeriol 7-O[2'-O-p-coumaroyl-6'-beta-O-acetyl-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside] named as ozturkosides A, B and C, respectively, were isolated, along with three known phenylethanoid glycosides, verbascoside, leucoseptoside A, martynoside and five known diterpenoids, 7-epicandicandiol, linearol, sidol, sideroxol, epoxyisolinearol. The structures were elucidated mainly by spectroscopic methods.     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Role of p53 and mismatch repair in PhIP-induced perturbations of the cell cycle

Heterocyclic amines, found ubiquitously in our diet, are carcinogenic and mutagenic. Among this class of compounds, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most abundant. To further understand the carcinogenesis of this compound, we studied the effects of PhIP on the progression of human lymphoblastoid cells through the cell-cycle. Cells differing in p53 or mismatch repair status were used to evaluate the role of those proteins. Following PhIP-treatment, a dose and time-dependent accumulation of p53 was found in cells containing functional p53. The augmentation of the p53 protein, accompanied by increases in p21-WAF1, confirms that the p53 is activated. The increase in p53 was independent of the mismatch repair status of the cells. Perturbations in the cell-cycle were also observed. Twenty-four hours after PhIP treatment, the activation of the G2-M checkpoint was evident. Functional p53 and mismatch repair were not required for the PhIP-induced G2-M arrest. The G2-M arrests were reversible and are interpreted as necessary for the repair of the PhIP-DNA lesions. Under treatment conditions where less than 5% of the cells survived, the G2-M arrests were absent.     

Optimizing antibody immobilization strategies for the construction of protein microarrays

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.     

High-yield expression and purification of human interferon alpha-1 in Pichia pastoris

For several years, interferon alpha-1, also known as interferon alpha-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves. Currently, recombinant human interferon alpha-D (rHuIFNalphaD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae. In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNalphaD using the Pichia pastoris system. The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNalphaD with better bioactivity than the commercially available rHuIFNalphaD molecule produced in E. coli.